Mesembryanthemum crystallinum L., a halophytic species, displays modified trichomes, epidermal bladder cells (EBC), on the surfaces of its aerial organs. EBCs serve to sequester excessive salt from underlying metabolically active tissues. To elucidate the molecular determinants governing EBC development in the common ice plant, we constructed a cDNA-based suppression subtractive hybridization library and identified genes differentially expressed between the wild-type and the EBC-less mutant. After hybridization, 38 clones were obtained. Among them, 24 clones had homology with plant genes of known functions, whose roles might not be directly related to EBC-morphology, while 14 clones were homologous to genes of unknown functions. After confirmation by northern blot analysis, 12 out of 14 clones of unknown functions were chosen for semi-quantitative RT-PCR analysis, and the results revealed that three clones designated as MW3, MW21, and MW31 preferentially expressed in the EBC-less mutant, whereas the other two designated as WM10 and WM28 preferentially expressed in the wild type. Among these genes, the expression of a putative jasmonate-induced gene, designated as WM28 was completely suppressed in the EBC-mutant. In addition, the deletion of C-box cis-acting element was found in the promoter region of WM28 in the EBC-less mutant. Overexpression of WM28 in Arabidopsis resulted in increased trichome number due to the upregulation of key trichome-related genes GLABRA1 (GL1), and GLABRA3 (GL3). These results demonstrate that WM28 can be an important factor responsible for EBC formation, and also suggest the similarity of developmental mechanism between trichome in Arabidopsis and EBC in common ice plant.

The common ice plant, Mesembryanthemum crystallinum L., a halophytic new functional leafy vegetable crop, develops epidermal bladder cells (EBCs) on the surfaces of its aerial organs. Our previous studies of the physiological characteristics of the wild-type and the EBC-less mutant indicated that EBCs sequester salt and maintain ion homeostasis within photosynthetically active tissues. The EBC has been thought to be a modified trichome; however, molecular mechanisms governing EBC development in the common ice plant have not been fully understood. Here, we have analyzed the steady-state mRNA abundance of nineteen cotton fiber-related gene homologs and eight Arabidopsis trichome development-related genes, and found that a MYB transcription factor homolog (McMYB2) and a GLABRA2-like gene (McC4HDZ) were preferentially expressed in wild-type plants, whereas a putative TRIPTYCHON (McTRY)-and CAPRICE-like gene (McCPC) were preferentially expressed in the EBC-mutant. The full-length cDNA sequences of these homologs were determined, and constructs containing McC4HDZ and McMYB2 were introduced into an Arabidopsis trichomeless mutant and wild-type plants. Overexpression of McMYB2 in wild-type Arabidopsis increased trichome number, associated with activation of the trichome development-related gene, GLABRA2 (GL2). Moreover, overexpression of McC4HDZ partially complemented trichome development in the trichome-less mutant of gl2-1, and resulted in increased trichome number in wild-type Arabidopsis, associated with the upregulation of key trichome-positive regulators GLABRA1 (GL1) and TRANSPARENT TESTA GLABRA1 (TTG1). These results suggest that McMYB2 and McC4HDZ could be functional in Arabidopsis trichome formation, implying that EBCs of the common ice plant and trichomes of Arabidopsis may share some molecular mechanisms in their development.Abbreviations: AGPC, acid guanidinium thiocyanate phenol chloroform; EBC, epidermal bladder cell; CAM, crassulacean acid metabolism; CaMV35S, cauliflower mosaic virus; CTAB, hecadecyltrimethylammonium bromide; RT-PCR, reverse transcription-PCR